: The CD45 family of transmembrane protein tyrosine phosphatases plays a key role in T cell activation by regulating the activity of Lck and Fyn protein tyrosine kinases. However, critical aspects of CD45 function remain unresolved. For example, CD45 is composed of multiple isoforms which differ only in their extracellular domains. These are differentially distributed on subsets of T cells having distinct functions and activation requirements. While the actual role of the individual isoforms remains unclear, it has been proposed that the CD45 extracellular domains differentially regulate signaling through the cytoplasmic domain. This raises the question as to whether CD45 has additional substrates. Moreover, the role of the two cytoplasmic domains in regulating enzyme activity and substrate specificity is unclear. Previous difficulties in expressing different individual CD45 isoforms in the same cell line have been overcome using a unique model whereby endogenous CD45 expression in the Jurkat human T cell leukemia line has been specifically blocked by transfection of a CD45-antisense gene. Such CD45- cells have been reconstituted to uniquely express either the largest o smallest human CD45 isoform, or various CD45 PTPase mutants. This model has allowed us to demonstrate CD45 isoform-specific differences in both IL-2 secretion and in the tyrosine phosphorylation of several key signaling moleules including zeta, ZAP-70, Vav, and SLP-76. Moreover, transfection of these CD45- cells with CD45 containing specific PTPase domain mutations (including substrate-trapping versions) has demonstrated that PTPase Domain 2 is critical for interaction of CD45 with several novel substrates including the TCR zeta chain, and is required for phosphorylatia n of ZAP-70. Based on our findings, this application seeks to provide new understanding of the role of CD45 in T cell activation. This will include defining the role of CD45 in regulating signaling through these new substrates and determining the mechanisms by which distinct CD45 isoforms differentially regulate T cell activation. In Aim 1 we will define the nature and consequences of the interaction of the CD45 cytoplasmic domain with these novel substrates. In Aim 2, we will determine how the expression of distinct CD45 extracellular domains corresponding to the largest (CD45(ABC ) and smallest (CD45(0)) isoforms results in altered capacity to produce IL-2; focusing on differential regulation of proximal events (including PTK activity and zeta phosphorylation) and Vav and SLP-76, that regulate key pathways involved in activation of the IL-2 promoter. These studies will provide fundamental knowledge about T cell activation signaling and growth control. As such, they should ultimately provide practical insight into hematological malignancie, immune-mediated diseases, and tolerance.